DESCRIPTION Neurotransmitters are released at the synapse by the fusion of synaptic vesicles with the plasma membrane. Insight into how neurotransmitters are released is of fundamental importance for understanding membrane fusion and brain function. Syntaxin 1 is a synaptic protein with an essential function in synaptic vesicle membrane fusion. At least eight synaptic proteins with which syntaxin 1 probably interacts during fusion have been identified. However it is unclear what syntaxin 1 exactly does during membrane fusion, and little is known about its native structure and mechanism of action. The current proposal describes experiments designed to address these questions. Four specific aims are proposed: 1. To determine the three-dimensional structure of syntaxin 1 by NMR spectroscopy. 2. To identify functional domains and interacting surfaces in syntaxin 1A by systematic mutagenesis and truncation mutants. The experiments will test the effects of mutations covering the entire structure of syntaxin 1A on its interactions with complexins, SNAP-25, a-SNAP, synaptobrevin, synaptotagmin, munc-18, munc-13, and Ca+ channels. 3. To elucidate the mechanisms of binding syntaxin 1A to target proteins by NMR spectroscopy. This will give insight into how residues identified in the second specific aim mediate specific binding. 4. To determine the biological significance of the interaction of syntaxin 1 with each target protein by mutating in transgenic mice, amino acid residues that are specifically essential for each interaction. These experiments will be based on results from the other specific aims and will be performed by homologous recombination in embryonic stem cells in mice. Together these experiments will allow insight into what syntaxins do during membrane fusion, how they do it, and what protein interactions are essential for doing it. The results will be important not only for our understanding of neurotransmitter release but also for membrane fusion in general. Finally, the results will aid in the design of therapies for diseases of the nervous system arising from defects in synaptic transmission.